REVIEW

Role of Transcription Factors in Differentiation and ￼￼￼
Reprogramming of Hematopoietic Cells
Hideaki Nakajima

Division of Hematology, Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan(Received for publication on September 29, 2010)(Revised for publication on November 22, 2010)(Accepted for publication on January 20, 2011)

Differentiation of hematopoietic cells is a sequential process of cell fate decision originating from hematopoietic stem cells (HSCs), allowing multi- or oligopotent progenitors to commit to certain lineages.
HSCs are cells that are able to self-renew and repopulate the marrow for the long term. They firstdifferentiate into multipotent progenitors (MPPs), which give rise to common lymphoid progenitors(CLPs) and common myeloid progenitors (CMPs). CMPs then differentiate into granulocyte monocyte
progenitors (GMPs) and megakaryocyte erythroid progenitors (MEPs), which are the precursors of
granulocytes/monocytes and erythrocytes/megakaryocytes, respectively. Lineage specification at differentiation branch points is dictated by the activation of lineage-specific transcription factors suchas C/EBPα, PU.1, and GATA-1. The role of these transcription factors is generally instructive, and theexpression of a single factor can often determine cell fate. Differentiation was long regarded as an irreversible process, and it was believed that somatic cells would not change their fate once they were differentiated. This paradigm was first challenged by the finding that ectopic cytokine signals could change
the fate of differentiation, probably through modulating internal transcription networks. Subsequently,
we and others showed that virtually all progenitors, including CLPs, CMPs, GMPs, and MEPs, stillretain differentiation plasticity, and they can be converted into lineages other than their own by ectopic activation of only a single lineage-specific transcription factor. These findings established a novel
paradigm for cellular differentiation and opened up an avenue for artificially manipulating cell fate forclinical use. (Keio J Med 60 (2) : 47–55, June 2011)

Keywords: transcription factor, hematopoiesis, differentiation, reprogramming, plasticity

￼￼￼Introduction

The hematopoietic system is undoubtedly the best-studied differentiation system throughout the body. Differentiation of hematopoietic cells originates from hematopoietic stem cells (HSCs), followed by a hierarchical processof cell fate decision and lineage commitment leading
to terminally differentiated mature blood cells.9 These
processes are often dictated by the instructive action ofhematopoietic transcription factors, which ultimately restrict the fate of differentiation.10 It was long thought thatcommitment was an irreversible process, and that cells
differentiated into a certain lineage could not change
their fate. However, this paradigm was first challenged
by the seminal work of Kondo et al. just over 10 years
ago, showing that ectopic cytokine signals could change

It has been only 5 years since the first report of successful generation of induced pluripotent stem (iPS) cells
by defined factors.1 Now we are in the decade of cellular
reprogramming,2–4 and people take it for granted that somatic cells of any type can be turned into an immaturestate by manipulation of transcription networks. Differentiation plasticity of somatic cells was actually proven ina hematopoietic system well before the generation of iPS
cells. We and others have reported lineage conversion ofhematopoietic progenitors as well as mature hematopoietic cells in response to certain stimuli such as ectopic
cytokine signals or activation of transcription factors.5–8

Reprint requests to: Hideaki Nakajima, MD, PhD, Division of Hematology, Department of Internal Medicine, School of Medicine,
Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan, E-mail: hnakajim@sc.itc.keio.ac.jp
Copyright © 2011 by The Keio Journal of Medicine.
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Nakajima H: Transcriptional Control of Hematopoiesis

the fate of differentiation.6 Since then, experimental evidence has accumulated supporting enormous plasticityof hematopoietic cells during differentiation, and a novel
paradigm has been reached that differentiation can becontrolled by modulation of transcription networks.
This review will focus on the differentiation pathwaysof hematopoiesis, the role of transcription factors in hematopoietic differentiation, and the amazing plasticity
retained by hematopoietic progenitors and mature blood
cells.
Differentiation Program of Hematopoietic Cells
Outstanding efforts by many groups over a decade have
now arrived at a comprehensive road map of hematopoietic differentiation from hematopoietic stem cells (HSCs)
to terminally differentiated cells.9,11,12 This was mostly
done by prospective isolation of HSCs and differentiationintermediates including hematopoietic progenitor cells(HPCs) using flow cytometry (FACS) (Fig. 1).
HSCs are cells that can self-renew and can produce alltypes of blood cells throughout life. The two critical features of HSCs that distinguish them from other hematopoietic cells are their self-renewal capacity and multilineage differentiation potential. In murine hematopoiesis,
HSCs make up a very small fraction of cells in the bonemarrow (BM) and lack lineage-affiliated cell surface
markers (Lin) but express high levels of c-Kit and Sca-1(KSL cells). The most primitive HSCs with self-renewal
and long-term repopulating (LTR) capacities (long-term
HSCs; LT-HSCs) are enriched in Thy1.1low/−, CD34low/−or CD150+ Flt3/Flk2− KSL.13–15 CD150+CD244−CD48−
cells have also been described as nearly pure HSCs.15
Wilson et al. have further shown that CD150+CD41−
CD48−CD34− KSL cells are more immature HSCs, asmall subset of which (about 20%) is in a dormant state.16
These populations of cells constitute about 0.01% of total
BM cells, and of this 0.01%, 1 in 3 to 1 in 5 cells were
found to have LTR capacity in a competitive repopulation
assay.
Cells with multilineage differentiation potential thatcan reconstitute the marrow for a limited period aretermed short-term HSCs (ST-HSCs), and are the immediate downstream progenies of LT-HSCs. ST-HSCs give rise
to multipotent progenitors (MPPs), cells with multilineage potential and a shorter reconstitution capability than
ST-HSCs. ST-HSCs and MPPs are considered to reside in
the CD34+ Flt3− KSL and CD34+ Flt3+ KSL fractions, respectively. However, other reports have stated that there
is no clear-cut separation between these two populations.
In fact, expression of Flt3 was reported recently to couple
with a loss of MegE potential, as described below.
Diversification of myeloid and lymphoid pathways follows after the MPP stage (Fig. 1A). Common lymphoidprogenitors (CLPs) are the most primitive common precursor for T and B lymphocytes and have a Lin− IL-7Ra+

Thy-1− Sca-1lo/− c-Kitlo phenotype.17 The hallmark of thispopulation is the expression of interleukin (IL)-7 receptor, which is critical for the expansion of the early lymphoid population. Indeed, abrogation of IL-7R leads tosevere combined immunodeficiency (SCID).
Common myeloid progenitors (CMPs), isolated as aLin− IL-7Rα− Sca-1− c-Kit+ FcgRII/IIIlo CD34+ population, are the earliest precursors for non-lymphoid cells thathave the potential for erythroid, megakaryocyte, granulocyte and monocyte development18 (Fig. 1A). CMPs further generate granulocyte-monocyte progenitors (GMPs;
Lin− IL-7Rα− Sca-1− c-Kit+ FcgRII/IIIhi CD34+) andmegakaryocyte-erythroid progenitors (MEPs; Lin− IL7Rα− Sca-1− c-Kit+ FcgRII/IIIlo CD34−), which give rise
to granulocytes/monocytes and erythrocytes/megakaryocytes, respectively18 (Fig. 1A). These progenitors wereshown to generate respective progenies in colony assays
and in in vivo transplantation experiments.
It was initially thought that bifurcation of the myeloid
vs. lymphoid lineage occurred right after the MPP stage,
but the story later turned out to be not as simple as that.
Recent evidence showing the existence of MPPs lacking megakaryocyte/erythroid (MegE) potential suggeststhat MPPs are heterogeneous.9,11,12 A current model of
myeloid/lymphoid commitment predicts that some MPPs
selectively lose MegE potential, leaving the lymphoidand granulocyte/monocyte potential intact, on their way
to becoming newly identified lymphoid primed multipotent progenitors (LMPPs) or granulocyte-monocytelymphoid progenitors (GMLPs) (Fig. 1B). LMPPs are described as CD34+MPPs expressing Flt3, and GMLPs are
CD34+MPPs marked by PU.1 expression. Of note, CMPs
are marked by the expression of GATA-1 as described
below. Please refer to the selected readings for a detailed
scheme of lineage commitment from HSCs.12
Transcriptional Control of Cell Fate Decision
It is widely known that lineage commitment of hematopoietic cells requires precise regulation of transcription
factors. In the past few decades, disruption of varioustranscription factors in the mouse genome has uncovereda fine picture of transcriptional networks regulating hematopoietic differentiation (Fig. 2).
Emergence of CMPs from MPPs is considered to depend on the simultaneous expression of PU.1 and GATA1. PU.1 and GATA-1 play critical, instructive roles for
granulocyte/monocyte (GM) and MegE lineage commitment, respectively.19–21 PU.1 transactivates a number ofGM and lymphoid genes, and PU.1-deficient mice display
profound defects in the development of B cells, monocytes and granulocytes.22 On the other hand, GATA-1
induces MegE-related genes, and is critical for the development of megakaryocytes and erythroid cells.23–27 A
counter-regulatory mechanism of PU.1 and GATA-1 activities has been reported in which either one of the twoKeio J Med 2011; 60 (2): 47–55

49

Fig. 1 Cellular pathways of hematopoietic differentiation.
(A) Classical myeloid versus lymphoid differentiation pathway. Myeloid and lymphoid pathways diverge right after the multipotent
progenitor (MPP) stage. (B) The new developmental pathways of myeloid versus lymphoid differentiation. This model integrates
newly identified lymphoid-primed multipotent progenitors (LMPPs) and granulocyte-monocyte-lymphoid progenitors (GMLPs), multipotent progenitors that selectively lost MegE potential. HSC, hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte-erythroid progenitor; GMP, granulocyte-monocyte progenitor.

factors represses the other’s expression or transcriptional
activity.28–31 A current model predicts that low-level expression of both PU.1 and GATA-1 in CMPs makes themcompetent for both GM and MegE lineages.32 Upregulation of PU.1 activity is considered to be a critical stepfor CMPs to commit to the GM lineage (and lose MegE
activity), whereas activation of GATA-1 skews CMPs tofollow the MegE pathway.33,34 The molecular mechanism
for this reciprocal regulation of PU.1 and GATA-1 is currently not well understood.
Differentiation of GMPs from CMPs depends on theexpression of CCAAT/enhancer binding protein (C/EBP)
α. C/EBPα-deficient mice lack mature neutrophils andeosinophils,35 and conditional disruption of C/EBPα inadult mice leads to the block of CMP-GMP transition.36
C/EBPα activates a number of myeloid genes, such as
granulocyte-colony stimulating factor receptor (G-CSF
R), macrophage colony stimulating factor receptor (MCSF R), myeloperoxidase (MPO), and neutrophil elastase.37–40 The early stage of neutrophil differentiation is
also regulated by Gfi-1.41–44 Gfi-1−/− mice have defects inneutrophil differentiation subsequent to the promyelocyte
stage. The terminal stage of neutrophil differentiation
critically depends on another member of the C/EBP fam-ily, C/EBPε. C/EBPε plays a critical role in the expressionof secondary and tertiary granule proteins, since lack of
C/EBPε leads to the neutrophil-specific granule deficiency phenotype.45
As discussed above, GATA-1 is critical for commitment
and further differentiation of the MegE lineage. The stem
cell leukemia (SCL) gene and EKLF are also essential forerythropoiesis, since lack of either protein leads to severe
anemia.46,47 GATA-1 collaborates with FOG-1 to induce
erythroid differentiation, whereas GATA-1 together with
AML-1 induces megakaryocytic differentiation.
Early lymphoid differentiation requires Ikaros.48 Commitment and further differentiation of CLPs to the B
lymphoid lineage critically depends on the expression of
three transcription factors: E2A, EBF and Pax5.49 Loss of
E2A or EBF leads to differentiation block at the pre-proB cell stage.49 In the case of T cells, GATA-3 is absolutely
essential for early thymocyte differentiation.50
Sequential Activation of Transcription Factors ￼￼￼Specifies Eosinophil and Basophil Commitment
It has been shown that mast cells, eosinophils and basophils are progenies of GMPs. Eosinophil-committed

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Fig. 2 Transcription factors regulating hematopoietic differentiation.
Transcription factors regulating lineage specification at various differentiation branch points or facilitating differentiation along specific lineage pathways are indicated.

progenitors (EoPs) can be isolated as cells with the IL5Ra+ Lin− Sca-1− CD34+ c-Kitlo phenotype.51 Mast cellprogenitors (MCPs) and basophil progenitors (BaPs)
have the Lin− CD34+ β7hi FcεRIαlo FcγRII/III+ c-Kitlo
Thy-1− and Lin− CD34+ FcεRIαhi c-Kit− phenotypes, respectively.52 It has been reported that a common precursor for mast cells and basophils, the basophil-mast cell
bipotent progenitor (BMCP), exists in spleen, and has theLin− CD13loCD34+β7 hi FcεRIα−FcγRII/III+c-Kit+Thy-1+
phenotype.53
Interestingly, the order of expression of C/EBPα and
GATA-2 specifies the eosinophil versus basophil lineage
from uncommitted progenitors54 (Fig. 3). If C/EBPα expression is followed by GATA-2, the cells become eosinophils; in turn, if the order of expression is reversed,
they become basophils. The same uncommitted progenitors can also generate neutrophil-committed progenitors and MCPs. However, sustained C/EBPα expression
is required for the cells to go into the neutrophil lineage
through GMP. In contrast, sustained GATA-2 expression
leads cells to become MCPs.

￼ Conversion of Hematopoietic Lineages by ￼￼￼
Ectopic Cytokine Signals
It was long believed that differentiation was an irreversible process and cells could not change lineages once they
were differentiated. However, this paradigm was recently
challenged by the finding of lineage infidelity of committed hematopoietic progenitors.6,55,56 A seminal reportfrom Kondo et al. showed that enforced signals frominterleukin (IL)-2 or granulocyte macrophage-colony
stimulating factor (GM-CSF) receptor could induce myeloid conversion in common lymphoid progenitor (CLP)and pro-T cells.6 When CLPs and pro T cells taken fromtransgenic mice expressing IL-2 receptor β chain were
cultured in the presence of IL-2, cells converted to granulocytes and monocytes after several days. Irreversible
commitment to myeloid lineage required at least 2 daysof cytokine stimulation. Interestingly, pro B cells andcommitted DN3 thymocytes were resistant to this IL-2induced lineage conversion.6,55 In addition, CLPs and pro
T cells could only be directed to GM lineages even undererythroid culture conditions. These data clearly showedthat at least CLPs and pro T cells possess latent myeloid
differentiation potential, which can be activated by ectopic cytokine signals.

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Fig. 3 Order of expression of C/EBPα and GATA-2 specifies eosinophil versus basophil lineage commitment.
In uncommitted progenitors such as CMPs, the expression of C/EBPα and GATA-2 is kept at a low level. If C/EBPα or GATA-2 isupregulated and maintained, cells are committed to neutrophil/monocyte or mast cell pathways, respectively. If C/EBPα is upregulated
first and GATA-2 follows, the cells become eosinophils. If the order of expression is reversed, they become basophils. EoP, eosinophilcommitted progenitor; BaP, basophil-committed progenitor; MCP, mast cell progenitors.

Ablation of Lineage-specific Transcription Factors
Unfixes Lineage Commitment
As described, commitment to a specific lineage critically depends on the activity of lineage-specific transcription factors. The process of commitment may require only transient action of transcription factors, but insome cases, their activity must be maintained throughout
differentiation to preserve lineage identity.57–59 One such
example is Pax5, an essential regulator for B cell differentiation (Fig. 4). Pax5 is critical for transition from prepro-B cells to pro-B cells, and its absence leads to differentiation arrest at the early pro-B cell stage. Interestingly,
Pax5−/− pro-B cells can be cultured indefinitely on stromalcells with IL-7. Furthermore, these cells are not actuallycommitted to B cells, but instead retained multilineage
differentiation potential.57,60 Conditional inactivation of
Pax5 in pro-B cells canceled their commitment to the B
cell lineage, and they regained their differentiation potential to macrophages and T cells.58 Moreover, conditional
ablation of Pax5 led to the dedifferentiation of mature B
cells back to early uncommitted progenitors that were capable of differentiating into T cells in vivo.59 These surprising observations underscore the critical role of Pax5
not only in commitment to the B cell lineage but also inmaintenance of B cell identity.
Another example came from the analysis of GATA1 knockout mice (Fig. 4). GATA-1−/− proerythroblasts
proliferate indefinitely on OP9 stroma in the presence oferythropoietin (Epo) and display differentiation potential
to myeloid and mast cells by the stimulation of granulocyte macrophage-colony stimulating factor (GM-CSF)
and interleukin (IL)-3, respectively.61 This observation
also suggests that lack of lineage-specific transcription
factors unfixes commitment to the respective lineage andallow cells to dedifferentiate back to an immature statethat retains multilineage differentiation potential.
Lineage Conversion by Ectopic Expression of ￼￼￼
Lineage-specific Transcription Factors
Lineage infidelity of differentiated hematopoietic cells
and strong lineage instructing capability of lineage-specific transcription factors suggested to us that such transcription factors may induce lineage conversion when
expressed ectopically (Fig. 4).
Iwasaki et al. were the first to show that enforced expression of lineage-specific transcription factor induced
transdifferentiation of hematopoietic progenitors.7 They
showed that ectopic expression of GATA-1 in CLPs, pro-

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Fig. 4 Lineage conversion and dedifferentiation of hematopoietic cells by modulation of transcription factors.
Lineage conversion of hematopoietic cells by activation of C/EBPα and GATA-1, and dedifferentiation of B or MegE cells by loss of
Pax5 or GATA-1. Reported pathways of conversion are shown by arrows. Loss of Pax5 or GATA-1 unfixes lineage commitment andleads to dedifferentiation back to multipotent progenitors.

B cells, CMPs and GMPs induced their transdifferentiation to the MegE lineage. Our group reported the conversion of CLPs, MEPs, early B cells and early T cells by
induction of C/EBPα activity.5 Likewise, retroviral expression of C/EBPα efficiently converted mature B cells,
CLPs and pro-T cells into the granulocyte/macrophagelineage.8,62
To investigate the conversion process in more detail,we generated transgenic mice expressing a conditional
form of C/EBPα (C/EBPα-ER) in all hematopoietic cells.5
Since activity of C/EBPα can be instantly induced inthese mice by addition of 4-hydroxy tamoxifen (4-HT),
this system allowed us to examine the detailed conversion process without cultivation of cells with cytokines
in vitro and infection of retrovirus, both of which may
artificially skew differentiation processes. When C/EBPα
activity was induced in MEPs and CLPs by addition of
4-HT, these cells were efficiently reprogrammed to theGM lineage. Clonal analysis revealed that the conversion
occurred at the single cell level. Interestingly, transient

activation of C/EBPα for 12 h for CLPs and 4 days for
MEPs was sufficient to initiate the conversion process.5
We have also shown that conversion to the GM lineage
could be induced not only in vitro, but also in vivo bysystemic administration of 4-HT.
Mechanism of Lineage Conversion
Given that lineage conversion is induced by the activation of transcription factors, it raises the question whether
the conversion process is accompanied by dedifferentiation of mature hematopoietic cells back to immatureprogenitors followed by tracing a differentiation path toother lineages, or whether cells are directly converted toother lineages through biphenotypic intermediate cells.
Analysis of C/EBPα-ER transgenic mice indicated thelatter possibility to be the case5 (Fig. 5). For example, we
identified cells expressing both erythroid (CD71) and GM(Gr-1, CD11b) markers during C/EBPα-mediated conversion of MEPs to the GM lineage. Similarly, conversion of

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53

Fig. 5 Mechanism of lineage conversion.
Conversion of various progenitors by ectopic activation of C/EBPα. Lineage conversion occurs through biphenotypic intermediate
cells, not by tracing back normal differentiation pathways. Cells in pink boxes are biphenotypic intermediate cells that have cell surface markers of original cells and myeloid lineage cells.

B cells to myeloid cells occurred through B220loCD11b+
or CD19+CD11b+ biphenotypic intermediate cells. It isinteresting to note that these intermediate cells have rearranged immunoglobulin (Ig) genes, clearly indicatingthat these cells originated from B cells. These observations demonstrate that enforced, ectopic expression oftranscription factors induces direct lineage conversion
between lineages through biphenotypic intermediate
cells.
In contrast, the dedifferentiation mechanism is responsible for the conversion of mature B cells to T cells in vivo
by inactivation of Pax559 (Fig. 4). As discussed in theprevious section, cells reacquire multipotentiality upon
loss of Pax5, and these cells can follow the physiologicaldifferentiation path to T cells. This is supported by thefact that Pax5-deleted mature B cells can restore T lymphopoiesis in Rag2−/− mice with a normal differentiation
pattern.59 Double-positive immature T cells taken fromthese mice retained the Ig rearrangement, indicating thatthe cells were derived from mature B cells.

Conclusions and Future Directions
It is now clear from in vitro studies that virtually all
hematopoietic cells at any stage of differentiation sustain
differentiation plasticity. The problem here is that most of
the studies were done in artificial settings, such as overexpression of transcription factors or ectopic cytokine
stimulation. This raises the question whether lineage conversion could occur in physiological settings in vivo. Our
study using transgenic mice expressing an inducible formof C/EBPα revealed that lineage conversion could occur
in the in vivo environment, at least by enforced activation of transcription factors. So the question is, are there
any physiological conditions where lineage-specific transcription factors are activated ectopically in vivo? Currently, we cannot think of any possibility of such a condition existing in vivo, suggesting that lineage conversion
is simply an observation indicating the latent plasticity ofhematopoietic cells. However, if in future studies novel
physiological pathways ectopically activating transcription factors were discovered, that development would

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Nakajima H: Transcriptional Control of Hematopoiesis

definitely add a novel and exciting twist to the current
understanding of hematopoiesis.
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